The present invention relates to a reagent which is useful in an immunodiagnostic assay method for determining the presence of an analyte in a fluid; to a method for producing the novel reagent; to an immunodiagnostic method using the novel reagent; and to a kit for use in carrying out the assay method.
There are known enzyme immunoassays for determining the presence of an agent in a biologic fluid. One such method is the so-called "EMIT" homogeneous enzyme immunoassay, in which a conjugate is produced comprising an antigen to be assayed and an enzyme. When the conjugate binds to the appropriate antibody, the activity of the enzyme decreases. Conversely, displacement of the conjugate by unbound antigen in the fluid sample results in an increase in enzyme activity which is proportional to the quantity of unbound antigen present. The EMIT procedure requires the use of small haptens, however, and is therefore unsuitable for assaying large molecules.
Pursuant to another method, the homogeneous enzyme-linked immunosorbent assay (ELISA), a solid-phase support, such as a plate, is sensitized by adsorption of antigen thereto. Test antibody in solution is then added, followed by addition of a ligand, which is typically an enzyme linked to a molecule specific for the bound antibody. Finally, a chromogen is added, which generates a colored end-product in the presence of the enzyme portion of the ligand. The optical density of the solution, measured at the end of a defined period, is proportional to the amount of enzyme present and thus to the amount of test antibody.
While the ELISA technique is fairly sensitive, it has the major drawback that it requires the use of solid supports and, consequently, requires washing steps after support sensitization as well as the addition of test antibody and ligand, respectively. All steps must also be performed consecutively. The time required for a typical ELISA is thus approximately 2 to 6 hours. Proper performance of an ELISA requires personnel who are knowledgeable concerning the properties of the assay. In addition, it has proved difficult to modulate the activity of enzyme conjugates of large antigens, such as proteins.
The enzyme immunochanneling assay (EICA) technique employs direct antigen-antibody binding to avoid the problem of modulating enzyme activity. A multienzyme complex is formed in which the product of one enzyme serves as a substrate for the next enzyme. The product can interact with the second enzyme before it escapes to the bulk solution. The first product is thus "channeled" to the second enzyme.
Those EICA methods which require use of solid-phase supports, such as beads or plates, have disadvantages similar to ELISA. While an EICA can be performed without solid supports, moreover, the adaptation of EICA to liquid is a difficult one. In order for the method to operate, the agent to be assayed usually must comprise two epitopes which are relatively close together. This proximity provides the microenvironment necessary for channeling. If a very large molecule has only two useful epitopes, and those epitopes are far apart, it may not be possible to create the required microenvironment. Furthermore, the EICA method requires a separate, specific antibody for each epitope. Since some molecules of potential diagnostic interest lack two distinct useful epitopes, competition between the two antibodies for the same epitope can occur. Under these conditions, it is very difficult to perform the EICA technique.
The channeling binding assay disclosed in U.S. Pat. No. 4,687,735 is carried out in a liquid medium without the use of plates, beads or other conventional solid supports at the start of the assay. But in the disclosed process a linking system is formed which incorporates one of the enzymes of the signal-producing system into a polymeric aggregate, producing a solid support in situ. As in a standard EICA, the analyte must have at least two distinct epitopes.